We have established and continuously cultured for 18 months allogeneic anti-H2-d CTL clones possessing a high degree of stable, specific lytic activity. Other cloned CTL have been selected for their loss of lytic activity. We plan to characterize the phenotypes of these functionally distinct clones using a panel of monoclonal antibodies (Mabs) and flow cytofluorometry. We will quantitatively analyze the expression of cell-surface molecules on the clones and correlate differences in expression with defects in CTL functions including motility, conjugate formation, and delivery of the lethal hit. Additionally, we will explore the functional role of LFA-1 and Lyt-2,3 in CTL killing by protecting determinants on these molecules during enzymatic proteolyses of the killer cells. By preincubating CTL with Mabs before treating with proteolytic enzymes, we can prevent removal of the Mab-bound determinant while proteolyzing nonprotected sensitive sites. Subsequently, the protective Mab will be dissociated from the determinant. Such a technique provides a potential means for mapping the functional determinant of the LFA-1 and Lyt-2,3 molecular complexes. For example, it may be possible to separate the role of Lyt-2 from Lyt-3 by Mab-protecting Lyt-3 and removing the Lyt-2 determinants. Finally, we are developing an assay for assessing CTL migration in response to possible chemotactic signals. We will determine if cloned CTL migrate in response to soluble factors including interleukin-2, products from target cells such as H-2 determinants shed from target cell membranes, and/or other possible, as yet unidentified, chemotactic molecules. (CS)